Non-heated samples were used as a reference. The solid-state properties of the mixtures were characterized by scanning electron microscopy, differential scanning calorimetry and X-ray diffraction. The drug release was studied in biorelevant media, including simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSIF) and fed-state simulated intestinal fluid (FeSIF). An enhanced apparent solubility and faster dissolution rate of NIF were observed in the heated mixture of NIF with CLAD-H in all tested biorelevant media , which was due to NIF amorphization in the high surface area nanocellulose powder. Ordinary MCC, which is essentially non-porous, did not produce an enhancement of a similar magnitude. The results of the study suggest that dry powder formulation using high surface area nanocellulose is a facile new strategy for formulating calcium channel blocker drugs, which could potentially be a viable alternative to currently used soft gel liquid capsules.Nifedipine (NIF) is a 1,4-dihydropyridine-based calcium channel blocker with poor solubility, whose bioavailability is highly dependent on the type of formulation. Dry powder mixtures of 20%  Nifedipine (NIF) is a BCS class II drug with solubility-limited bioavailability. Furthermore, it experiences substantial first-pass metabolism in liver 42–56%). It is mainly used to treat cardiovascular diseases, such as hypertensive crisis, angina pectoris and chronic hypertension . The pha–56%. It max for SGCs showed high variability, with values in the range of 0.25–2.9 h having been reported in other studies [max and AUC between SGCs and film-coated tablets. According to the biowaiver monograph for NIF IR formulations, NIF is not ionized in the pH range found in GIT and thus, its solubility is independent of the pH of the medium in this range . After o studies . NormallIn order to develop solid-dosage form alternatives to SGCs in the form of tablets, the issue of low NIF solubility needs to be addressed. Several strategies for improving the solubility of NIF have been employed in the past, including micronization , nanocry2/g, but nanocellulose can provide much larger surface area, i.e., 60–100 m2/g [MCC is a common excipient, which is used as a binder/diluent in tablets and capsules both by wet granulation and direct compression. Ordinary MCC is characterized by a relatively low surface area, i.e., ≤1 m100 m2/g ,22. Furt100 m2/g  and it c100 m2/g . Nanocel100 m2/g ,26,27.It has previously been shown that the dissolution rate of NIF can be significantly increased (30–37-fold) by hot-melt extrusion of multi-component mixtures with polyvinylpyrrolidone (PVP) or hydroxypropylcellulose (HPC), i.e., NIF-PVP-MCC or NIF-HPC-MCC, which was due to crystalline-to-amorphous drug transitions . It shouWe have recently shown that the formulation of ibuprofen and other arylpropionic acid derivatives with nanocellulose can result in enhanced solubility and rapid dissolution rate due to amorphization of the drug . OrdinarCLAD was provided by FMC Biopolymers . MCC (Avicel PH101) and nifedipine (NIF) was purchased from Sigma Aldrich  (≥98% purity). Biorelevant media of simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSIF) and fed-state simulated intestinal fluid (FeSIF) were prepared using powder purchased from biorelevant.com  according to the manufacturer’s instructions. All chemicals were used as received without further purification. Since nifedipine is a photosensitive substance, all mixtures were placed in amber vials, which were additionally covered in aluminum foil for extra photoprotection. The weight ratio between NIF and cellulose was 2:8. Typically, 20 mg of NIF was mixed with 80 mg of cellulose in a sealed glass vial with cap using a Turbula mixer  for 15 min at 72 rpm.Following the preparation of the powder mixtures as described above, the sealed vials were heated to the melting temperature of NIF  for 1 h. It was ascertained that no thermal degradation of NIF occurred during the heat treatment, which is shown in −2 mbar and 35 mA and for a sputtering time of 30 s. The Zeiss Merlin FEG-SEM instrument  was used. Powder samples were mounted on aluminum stubs using double-adhesive carbon tape and further sputtered with Au/Pd to reduce charging effects. A sputter coater  was used. The sputtering settings were 4 × 10The DSC measurements were performed with the DSC 3+ instrument . Nitrogen gas at a flow rate of 60 mL/min was utilized throughout the measurements. −1 before being heated to around 283.15 K (10 °C) at a heating rate of 0.7 K min−1. All measurements were obtained in triplicate.A total of 10 mg of cellulose was placed in 1.5-mL Eppendorf tubes. One milligram of de-ionized water was added and the tube was shaken rigorously. After that, the tube was left overnight at room temperature. On the next day, the tube was centrifuged at 20,000 rpm for 6 min. Water was decanted and any free drops were carefully removed with a piece of paper. The samples were placed in aluminum pans with a lid and first cooled from room temperature to 243.15 K (−30 °C) at a rate of 10 K minpr is the radius of pore (nm) and ΔT is the difference between the peak maximum for melting of pore-confined water and peak value for melting of bulk water.The pore size was calculated according to Landry :(1)rp= before being heated to around 473.15 K (200 °C) at a heating rate of 10 K minCrI) of NIF was calculated as follows:mixH is the enthalpy of NIF melting in the mixture, ΔNIFH is the melting enthalpy of pure NIF and a is the correction factor corresponding to NIF content, i.e., a = 1 for pure NIF and a = 0.2 for 20% w/w mixture. The crystallinity index  with Bragg-Brentano geometry  was used. The operating current settings were 40 kV and 40 mA. The 2θ angle was varied between 10 and 45° at 0.02° scan steps. The data were collected on flat powders, which were placed in reduced background specimen holders supplied by the manufacturer (Bruker).The dissolution test was performed in SOTAX  apparatus using 500 mL of the biorelevant medium per dissolution vessel. The dissolution bath was covered with aluminum foil for extra photoprotection. The temperature for each dissolution vessel was maintained at 37 °C. The same amount of mixed samples was used in all timings and all different media (20 mg NIF + 80 mg cellulose). Each group of non-heated and heated samples was run in triplicate. The paddle speed for each dissolution vessel was 50 rpm. The sampling times for all media were 5, 15, 30, 45, 60, 90 and 180 min, respectively. At each time point, 5 mL of the medium was withdrawn and filtered through a 0.45-μm PTFE filter after discarding the first 2–3 mL. A total of 1.5 ml of the remaining sample was transferred in glass vials for further analysis by HPLC.The HPLC-UV system was optimized for the detection of NIF prior to the analysis. The liquid chromatography system used was a quaternary pump  with an autosampler and Xbridge BEH C18, 3.5 μm, 2.1 × 50 mm, Waters column. The column temperature was 50 °C and injector temperature was 20 °C. The mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 0.8 mL/min. The injector wash was 50% acetonitrile. The retention time was 4.45 min and run time was 8.0 min. UV detection at the wavelength of 343 nm was used. Calibration samples were run prior to the analysis of the study samples by preparing the NIF stock solution in the biorelevant medium at a concentration of 250 μg/mL. The stock solution was used as the calibration standard by a subsequent gradual dilution of 1:250. The normal and heated powder mixtures of NIF-CLAD were produced as described above and stored at 40 °C and 75% RH in a desiccator, which was equilibrated over a saturated NaCl solution. The solid-state stability of the mixtures was regularly monitored using the DSC instrument  to record melting enthalpies. The DSC procedure was performed as described above. Prior to discussion of the observed results, it is important to note that the term amorphization that is used throughout the rest of the text is meant to indicate a loss of long-range periodicity that is an important characteristic of fully crystalline structures, while some short-range order may still be present.As shown in When studying the XRD profiles of the mixtures, we found that the characteristic sharp peaks for crystalline NIF are clearly seen in both non-heated and heated mixtures of MCC, which are overlaid on the cellulose pattern. These sharp peaks of NIF are also visible in the non-heated mixture of NIF-CLAD. However, they are essentially suppressed in the heated NIF-CLAD mixture. The absence of the characteristic sharp diffraction peaks of NIF suggests that the drug is in the amorphous state, which is consistent with DSC observations. Overall, the results of the DSC and XRD analysis suggest that the molecular state of NIF in the heated mixture with CLAD is substantially different from that of the non-heated CLAD mixture as well as those for non-heated and heated mixtures of MCC. The experimental evidence from two independent solid-state characterization methods suggests that NIF is in the amorphous state in the NIF-CLAD-H mixtures, which is expected to result in a higher dissolution rate.max of NIF release from NIF-CLAD-N mixture in SGF was relatively higher than that of the NIF-MCC-H sample, leveling at 7 ± 1 μg/mL after 90 min. However, the initial rate of NIF dissolution during the first 15 min for NIF-CLAD-N was not as rapid as that for the NIF-MCC-H mixture and it was more similar to that of NIF-MCC-N. The release of NIF from the heated CLAD mixture showed an interesting profile. The amount of released drug for NIF-CLAD-H sample was almost 2 times higher after 5 min and 2.7 times higher after 45 min than that for NIF-MCC-H at similar time points. Compared to the non-heated MCC and CLAD mixtures, after 5 min, the release of NIF from NIF-CLAD-H was nearly 20 times more NIF. After 45 min, a Cmax value of 16 ± 1 μg/mL was achieved in NIF-CLAD-H after which it gradually decreased to 14 ± 2 μg/mL, where it remained for the rest of experiment. Apparently, NIF remained in the supersaturated state throughout the entire experiment. It should be noted that the Cmax for NIF-CLAD-H in SGF corresponds to 40% of the total loaded dose.In order to investigate the dissolution behavior of NIF in mixtures with cellulose, in vitro dissolution tests in biorelevant media were performed. max in FaSIF for NIF-MCC-N and NIF-MCC-H was 7.7 ± 0.5 and 12.4 ± 2.4 μg/mL, respectively. On the other hand, the Cmax in FaSIF for NIF-CLAD-N and NIF-CLAD-H was 6.6 ± 1 and 25.6 ± 1.7 μg/mL, respectively. Thus, the Cmax in FaSIF for NIF-CLAD-H was two times higher than that for NIF-MCC-H. Furthermore, this high amount of dissolved NIF was achieved within the first 15 min of dissolution in FaSIF for NIF-CLAD-H, which is nearly 7.7 times higher than that for NIF-CLAD-N sample at a similar time point. It should also be noted that even at the highest level of dissolution observed in the NIF-CLAD-H sample, the amount of released NIF was only 64% of the total loaded dose.max in FeSIF for NIF-MCC-N and NIF-MCC-H was 16.4 ± 1.7 and 15.3 ± 2.3 μg/mL, respectively. On the other hand, the Cmax in FeSIF for NIF-CLAD-N and NIF-CLAD-H was 11.7 ± 0.5 and 28.3 ± 0.9 μg/mL, respectively. Thus, the Cmax in FeSIF for NIF-CLAD-H was 1.8 times higher than that for NIF-MCC-H, which is comparable to that in FaSIF. Furthermore, this high amount of dissolved NIF was achieved within the first 5 min of dissolution in FeSIF for NIF-CLAD-H, after which it fluctuated between 25 and 28 μg/mL for the rest of the experiment. The amount of NIF dissolved during the first 5 min of the experiment was nearly 6 times higher than that for NIF-CLAD-N mixture at a similar time point. It should also be noted that even at the highest level of dissolution observed in the NIF-CLAD-H sample, the amount of released NIF was only 70% of the total loaded dose.Overall, there seems to be a significant improvement in the apparent solubility and dissolution properties of NIF in heated CLAD mixtures in all tested biorelevant media due to amorphization of the drug.In order to confirm that NIF remains amorphous in NIF-CLAD-H mixture over a sufficiently long period of time, solid-state stability studies were performed by storing the NIF-CLAD-N and NIF-CLAD-H mixtures at 40 °C and 75% RH. In this work, we showed an enhancement of the solubility and dissolution rate of NIF with heated CLAD mixtures. NIF supersaturation was achieved, which remained relatively stable for at least 3 h. For amorphous solid dispersions, it is common to observe the so-called spring-and-parachute effect in which the solubility of the drug rapidly reaches transient supersaturation level before gradually declining to the thermodynamically stable equilibrium solubility level. Because the supersaturated solubility of NIF remained relatively stable over the period of experiment, it could be speculated that CLAD has the ability to prevent the re-crystallization of NIF. However, this effect requires further investigation. The amorphization of NIF upon mixing and heating with high surface area nanocellulose is associated with the observed effect. A similar effect was previously described for NSAIDs from profens class , which sIt has been reported in literature that NIF shows pH-independent solubility at the physiological pH of GIT. In this work, we observed that NIF solubility in SGF was lower than that in FaSIF and FeSIF. However, it should be noted that the pH-independent solubility data of NIF has mainly be obtained for SGCs at various pH values and there is a limited number of available articles that focus on its dissolution in various buffer solutions . It shoumax was much lower and tmax was longer than expected for the highest dose, i.e., 20 mg [max and the decelerated elimination phase [max and tmax values compared to commercially available NIF SGCs for bioequivalence. If the new dosage form is not bioequivalent to SGCs, this may lead to concentrations either below or above the recommended therapeutic window. Thus, since the BCS-based biowaiver procedure for NIF cannot be recommended for either the SGC or tablet IR formulations, bioequivalence needs to be ensured in vivo [The facile method of IR NIF formulation with nanocellulose opens new possibilities for designing directly compressed tablets with enhanced PK properties as an alternative to traditional SGCs. It has been reported that IR NIF SCGs experience a deviation from a linear correlation between AUCs and doses at a higher drug content, i.e., 20 mg vs. 5–10 mg. In particular, the mean dose-normalized C., 20 mg . Accordion phase . Thus, tAn enhanced solubility and faster dissolution rate of NIF were observed in NIF-CLAD-H mixtures in all tested biorelevant media, i.e., SGF, FaSIF and FeSIF. The observed enhancement of solubility and dissolution was due to amorphization of NIF in high surface area nanocellulose powder. Ordinary MCC failed to produce an enhancement of a similar magnitude. Amorphous NIF-CLAD-H mixtures were stable under 40 °C and 75% RH conditions for at least 54 days, when the study was halted. The results of the study suggest that dry powder formulation using high surface area nanocellulose with heat is a facile new strategy for formulating calcium channel blocker drugs, which could be an alternative to currently used SGCs. Future studies should focus on determining their bioequivalence to NIF SGCs to further elucidate the clinical potential of the platform.


Candida albicans. The antifungal therapy represents the standard of care but due to the high costs of treatment and to the inability to prevent recurrences, the development of alternative therapeutic approaches is much-awaited. Recently, we have shown that the pathogenesis of C. albicans in the gut is modulated by IL-9, a pleiotropic cytokine able to promote both inflammation and tolerance during C. albicans infection. Herein, by using a mouse model of VVC, we similarly demonstrated that IL-9 might exert a dual role in VVC by contributing to inflammation during the initial immune activation and promoting resolution thereafter. Specifically, IL-9 has a pro-inflammatory activity at the onset of VVC by promoting NLRP3 inflammasome activity and mucosal mast cells expansion but a tolerogenic role in the resolution phase by promoting IL-1Ra production and connective tissue mast cells activation. We further show that a timely IL-9 neutralization at the onset of the inflammatory response ameliorated symptoms and vaginal pathology. Given that vaginal fluids from patients with recurrent VVC had higher levels of IL-9, these findings, by providing novel insights into the pathogenesis of VVC, may pave the way for alternative therapeutic strategies based on IL-9 neutralization.Vulvovaginal candidiasis (VVC) is a common fungal infection caused by  Candida albicans, an opportunistic fungus commonly present in human mucosal surfaces, is pivotal to limit inflammation and maintain homeostasis. Vulvovaginal candidiasis (VVC) is the most common mucosal fungal infection that significantly affects the quality of life, particularly in its chronic and recurrent forms (RVVC)  , 2. The s (RVVC) .C. albicans to switch from commensal to pathogen in the gastrointestinal tract . Murine experiments were performed according to the Italian Approved Animal Welfare Authorization 360/2015-PR and Legislative decree 26/2014 regarding the animal license obtained by the Italian Ministry of Health lasting for 5 years (2015–2020).Female C57BL/6, 8–10 weeks for age, were purchased from Charles River. 6 viable C. albicans 3153A blastospores from early-stationary-phase cultures. Murine monoclonal anti-IL-9 antibody or control isotype IgG were administered intraperitoneally at the dose of 10 mg/kg starting the day of infection. Mice were sacrificed at different time points. CFUs were enumerated after incubation of Sabouraud-dextrose agar plates at 36°C for 24 h and expressed as Log CFU/100 ml of vaginal fluid. Cytospin preparations of the lavage fluids were stained with May-Grünwald-Giemsa.Estrogen-treated mice were inoculated intravaginally with 5 × 10For histology, paraffin-embedded tissue sections (3–4 micrometer) of the vagina were stained with periodic acid-Schiff. For immunofluorescence, vaginal sections were incubated at 4°C with anti-NLRP3 and anti-pNLRC4 antibodies followed by secondary TRITC or FITC antibodies, respectively. The sections were observed using the BX51 microscope equipped with a high-resolution DP71 camera.Il9, TGACCAGCTGCTTGTGTCTC and GTGGCATTGGTCAGCTGTAA; Il1Ra, TTGTGCCAAGTCTGGAGATG and CAGCTGACTCAAAGCTGGTG. The PCR primers for the other genes were as described . Cells were lysed and total RNA was reverse transcribed with cDNA Synthesis Kit (BioRad), according to the manufacturer's instructions. The PCR primers sequences (5′-3′) were as follows: escribed . The levt-test, one- or two-way ANOVA with Bonferroni post-hoc test were used to determine the statistical significance. Significance was defined as p < 0.05. Data are pooled results (mean ±SEM) or representative images from three experiments. GraphPad Prism software 6.01 (GraphPad Software) was used for analysis.Student's Il9R−/− mice intravaginally infected with Candida blastospores and first evaluated the expression of Il9 in vaginal tissue. As shown in Figure Il9 increased early and was observed throughout the infection in C57BL/6 mice, but was unaffected in Il9R−/− mice, a finding suggesting that IL-9 expression is IL-9R-dependent. We next evaluated the susceptibility of Il9R−/− mice to vaginal candidiasis. As compared to C57BL/6, Il9R−/− mice showed a reduced inflammation in the initial phase of infection, but an increased susceptibility in a later phase, as indicated by fungal burden  in the inflammatory phase, while promotes the activity of tolerogenic connective tissue MC (CTMC) in the resolution phase . To evalr Figure . As expey Figure . Given tn Figure  and restn Figure .Finally, we assessed the translatability of the results obtained in the murine model to human VVC. In agreement with previous data, the vaginal fluids of patients with recurrent VVC (RVVC) had lower levels of IL-1RA and higher amounts of IL-1β than healthy controls Figure . In addiC. albicans commensalism and pathogenicity  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University Ethics Committee of Perugia (Prot. 2012-028).in vivo experiments. J-CR provided the knockout mice. PM, AB, LR, CC, and GR analyzed the data and wrote the paper.GR and VO designed the experiments. MB performed immunofluorescence and most of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SiCaOC with high Ca content  exhibits the presence of crystalline calcium silicate (mainly pseudowollastonite). Furthermore, it has been shown that the incorporation of Ca into the SiOC glassy network has a significant effect on its porosity and specific surface area. Thus, the as-prepared Ca-free SiOC material is shown to be non-porous and having a specific surface area (SSA) of 22.5 m2/g; whereas SiCaOC with Ca/Si molar ratio of 0.05 exhibits mesoporosity and a SSA value of 123.4 m2/g. The further increase of Ca content leads to a decrease of the SSA and the generation of macroporosity in SiCaOC; thus, SiCaOC with Ca/Si molar ratio of 0.12 is macroporous and exhibits a SSA value of 39.5 m2/g. Bioactivity assessment in simulated body fluid (SBF) confirms the hydroxyapatite formation on all SiCaOC samples after seven days soaking, unlike the relatively inert ternary silicon oxycarbide reference. In particular, SiCaOC with a Ca/Si molar ratio of 0.05 shows an increased apatite forming ability compared to that of SiCaOC with Ca/Si molar ratio of 0.12; this difference is considered to be a direct consequence of the significantly higher SSA of the sample with the Ca/Si ratio of 0.05. The present work indicates two effects of Ca incorporation into the silicon oxycarbide glassy network on its bioactivity: Firstly, Ca2+ is shown to contribute to the slight depolymerization of the network, which clearly triggers the hydroxyapatite formation (compare the bioactive behavior of SiOC to that of SiCaOC with Ca/Si molar ratio 0.12 upon SBF exposure); secondly, the Ca2+ incorporation seems to strongly affect the porosity and SSA in the prepared SiCaOC materials. There is an optimum of Ca loading into the silicon oxycarbide glassy network (at a Ca/Si molar ration of 0.05), which provides mesoporosity and reaches maximum SSA, both highly beneficial for the bioactive behavior of the materials. An increase of the Ca loading leads, in addition to the crystallization of calcium silicates, to a coarsening of the pores  and a significant decrease of the SSA, both negatively affecting the bioactivity.In the present work, Ca-containing silicon oxycarbides (SiCaOC) with varying Ca content have been synthesized via sol-gel processing and thermal treatment in inert gas atmosphere (pyrolysis). It has been shown that the as-prepared SiCaOC materials with low Ca loadings (Ca/Si molar ratios = 0.05 or 0.12) were X-ray amorphous; their glassy network contains Q Silicon oxycarbide (SiOC) has high biocompatibility ,2,3 and 2/g) .,53.52,53A comparison between SiCa5-T and SiCa12-T shows more active apatite formation on SiCa5-T than on SiCa12-T: Hydroxyapatite grows on SiCa5-T to a size of several micrometers; whereas, hydroxyapatite clusters on SiCa12-T are much smaller (~0.5 um). Furthermore, the higher HA formation on SiCa5-T is also confirmed by FTIR results see a.3 fraction) and lower network carbon , SiCa5 is amorphous after 1100 °C pyrolysis. This high crystallization resistance is desirable if thermal processing of the material is anticipated.Calcium nitrate-based sol-gel synthesis proves to be applicable for preparing Ca-containing silicon oxycarbide materials (SiCaOC). The porosity of synthesized SiCaOC is shown to be tunable depending on the content of calcium nitrate: (i) Calcium nitrate precipitation from pore liquids during gel drying is considered to induce both anti-shrinkage and pore-filling effects, resulting in mesoporous xerogel at a low content (Ca/Si = 0.05) and microporous xerogel at a high content (Ca/Si = 0.12 or 0.50); (ii) the decomposition of clustered calcium nitrate precipitated in xerogel during polymer-to-ceramic transformation is correlated to the formation of macropores at Ca/Si = 0.12, while higher loading with Ca/Si = 0.50 leads to the formation of crystalline calcium silicate (pseudowollastonite), the good sinterability of which leads to pore closure at pyrolysis temperature. The mesopores of xerogel at Ca/Si = 0.05 show a high resistance against shrinkage and collapse at pyrolysis temperature, leading to mesoporous SiCaOC.3 sites, high specific surface area in the case of mesoporous SiCaOC is shown to improve apatite forming ability significantly. Thus, the facile approach for preparing SiCaOC in the present study has achieved two bioactivity-enhancing effects simultaneously . The combination of mesoporosity, crystallization resistance, bioactivity and biocompatibility makes the investigated mesoporous SiCaOC suitable for potential bone tissue engineering applications.SBF assessment confirms apatite forming ability of all SiCaOC materials. Besides the network depolymerization due to Ca incorporation at Q


The release profiles indicated that SN-38 presented an initial burst release within 12 h, and sBSANPs had a slow release pattern. Flow cytometry results showed that the fluorescence intensity of sBSANPs was significantly higher than that of the control group. The confocal images also confirmed that sBSANPs were taken up by MDA-MB-468 cells. Moreover, we found that a larger BSANP size resulted in an increased hemolytic effect. In vivo animal studies demonstrated that loading of SN-38 into bovine serum albumin nanoparticles could minimize the initial concentration without extending the elimination half-life, but significantly minimized the Cmax (p < 0.001) as compared with irinotecan treatment.Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with a worse prognosis than other types. There are currently no specific approved treatments for TNBC. Albumin is a promising biomimetic material that may be fabricated into nanoparticles to possibly exert passive effects on targeted tumors. Irinotecan has been extensively used in clinical settings, although a high dosage is required due to its low efficiency of conversion into the active metabolite SN-38, also known as 7-ethyl-10-hydroxy-camptothecin. The aim of this work was to optimize SN-38-loaded bovine serum albumin nanoparticles (sBSANPs) and evaluate their potency against TNBC. The sBSANPs were characterized by a small size of about 134–264 nm, a negative charge of −37 to −40 mV, an entrapment efficiency of 59–71%, and a particle yield of 65–86%. The cytotoxicity assays using sBSANPs showed a higher potency specifically against both MDA-MB-468 and MDA-MB-231 cells  compared to MCF-7 , and exhibited an extremely low IC HER2 expression. Individuals with TNBC have been found to have worse overall survival (OS) when compared with individuals with ER+/HER2−breast cancers, and the difference was found to be most pronounced in the first two years by a large observational prospective study  is the concentration of SN-38.The preparation of BSA solutions with SN-38 for the fluorescence quenching study was identical to the preparation of sBSANP. The fluorescence spectrophotometer was a model F-4500 . The excitation wavelength was 280 nm (slit = 2.5 nm) and the emission wavelength was 300–700 nm (slit = 10 nm). The samples were kept in dry bath incubators at 25, 30, and 37 °C prior to measurement. Fluorescence quenching can be described using the Stern–Volmer Equation (3) :(3)I0I=1−1 at a resolution of 1 cm−1.The samples were diluted with approximately 1% KBr mixing powder and pressed into 16 mm disks at 15 t for analysis using a Fourier-transform infrared (FTIR) spectrometer . The wavenumbers ranged from 4000–380 cmv/v)) at pH 7.4, and stirred at 100 rpm at a constant temperature of 37 °C. The receptor medium was sampled at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h and analyzed by HPLC. The samples were immediately supplemented with an equal volume of fresh receptor medium after each sampling. The samples in this study included sBSANP-F75, -F62.5, and -F40, and the SN-38 solution. The solvent for SN-38 was the receptor medium and the concentration was the theoretical concentration of sBSANPs (0.5 mg/mL). Release profiles and correlation coefficients of SN38 free solution and SN38-loaded sBSANPs were investigated, as shown in A total of 1 mL of the sample was placed in dialysis tubing (MWCO = 6000–8000 kDa). Each dialysis bag was fully submerged in 5 mL receptor medium, which comprised PBS with 99% ethanol (1:1 (v/v)) as the stock solution and stored at 4 °C for later use. To analyze hemolysis, 100 µL stock solutions were mixed with 50 µL sBSANP or BSANP formulations along with 2.45 mL PBS (pH 7.4). The mixture was incubated at 37 °C under constant shaking for 1 h, and centrifuged at 2000 RCF for 5 min to separate the hemoglobin from cell debris. Finally, the supernatant was assayed at 415 nm using a UV spectrometer. The measured value for the positive control was RBCs dissolved in water and was taken as 100% hemolysis, while the value for the negative control was RBCs dissolved in PBS (pH 7.4) and was taken as 0% hemolysis 2 term in the Stern–Volmer Equation (5):aK denotes the association constant of the quencher–fluorophore complex [Quenching mechanisms are usually classified as dynamic, static, or both. Dynamic quenching, also known as collisional quenching, is defined as when the quencher assists the fluorophore in returning to the ground state without proton emissions upon contact with the fluorophore; since the collision of the quencher to fluorophore depends on diffusion, the ncreases . On one ncreases , since t complex . The othsvK value increased with temperature, indicating dynamic quenching. The results also showed that the BSA solution with SN-38 after probe sonication had a combination of dynamic and static quenching because the plot showed upward curves, and the svK value decreased as the temperature rose. We deduced that the probe sonication not only dissolved the SN-38 crystals, but facilitated the formation of the quencher–fluorophore complex. In other words, probe sonication might have enhanced the drug–albumin affinity.FTIR is a useful tool for evaluation of the surface chemistry of particles after modification and the change in the secondary structure of proteins ,35. FiguAdditionally, the amide I/II ratio can be used to evaluate the secondary structure of proteins during denaturation. This ratio increases as the protein undergoes denaturation, and decreases as the peptides become increasingly ordered structures . Here, the result was as expected: the ratio of the nanoparticles was higher than that of the BSA lyophilized powder .The release models of the three formulations fit the zero-order and Hixson–Crowell model best among the five different mathematical models . The gooThe fit of the release models was calculated using the data from 0 to 12 h, because the results showed good linearity within this range, and lost linearity after 12 h. This loss of linearity was expected, since it has been found in every reported release test using dialysis membranes. This trend was also observed for the control. Therefore, we did not consider that the loss of linearity was due to the formulation design, and should instead be viewed as a limitation of the dialysis membrane.Guidance for Industry Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients [The hemolysis assay is a useful tool for conducting a preliminary evaluation of the toxicity of pharmaceutical excipients. Exposure to foreign objects causes hemolysis. Due to genetic variation, some individuals are more vulnerable to hemolysis, even at low concentrations. Having received growing attention, the hemolysis assay has also been recommended by the FDA in evaluating the hemolytic potential of IV route materials in the cipients . The hemolysis–nanoparticle relationship has been reported in several studies ,39. The Three cell lines were used in this study. The cell lines MDA-MB-468 and MDA-MB-231 are basalA and B-like triple-negative breast cancer cell lines, and the basal-like subtype accounts for 70% of TNBC cases , whereas50 values in MDA-MB-231 and MDA-MB-468 were 5- to10-fold lower than that for MCF-7. This suggests that MDA-MB-468 (TNBC) was more sensitive to SN-38 cytotoxicity than MCF-7 (non-TNBC). The IC50 values of the three sBSANP formulations matched the IC50 of SN-38 solution in the MDA-MB-231, MDA-MB-468, and MCF-7 cell lines, suggesting that the preparation of sBSANPs did not alter its anticancer effect toward cancer cell lines. These IC50 values were also in good agreement with previously reported results by Goldenberg et al. (2015) [In . (2015) .50 plots neither demonstrated sigmoidal curves . This could be explained by (1) the crosslinked albumin nanoparticles being immediately recognized by scavenger receptors and removed from the circulatory system; (2) the plasma sample preparation being unable to extract SN-38 within the albumin nanoparticles; and (3) the entrapment efficiency being overestimated by setting The concentration of SN-38 in vehicle was equivalent to sBSANP-F62.5 (0.315 mg/mL). In This study addressed the preparation, characteristics, safety efficacy, and preliminary evaluation of the pharmacokinetic behavior of SN-38-entrapped albumin nanoparticles. From the in vitro experiments, the results demonstrated that the application of sBSANPs has potential as a safe approach for TNBC treatment. In the pharmacokinetic evaluation, although sBSANPs could not elongate the elimination half-life, these nanoparticles were capable of influencing pharmacokinetic parameters. To understand whether sBSANPs accumulate at tumor sites, a breast cancer xenograft model is required, and this study will be underway in the near future.


Lactobacillus plantarum (L. plantarum) containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup J Avian Leukosis Virus (ALV-J) infection were assessed. In the current study, the gp85 protein of ALV-J was expressed on the surface of L. plantarum with the surface-display motif, pgsA, by constructing a shuttle vector pMG36e:pgsA:gp85. Surface localization of the fusion protein was verified by western blotting and flow cytometry. Subsequently, Specific Pathogen Free Hy-Line Brown layer chickens were orally vaccinated with the recombinant L. plantarum and presented with high levels of serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) titers in bile and duodenal-mucosal fluid. After challenged with ALV-J of a 3 × 103 50% tissue culture infective dose (TCID50), serum samples of the chickens were collected and viremia was analyzed. Results showed that, compared to the L. plantarum and PBS control group, the recombinant L. plantarum group showed a significant rise in antibody levels after inoculation, and provide improved protection against ALV-J according to viremia detection. These results indicate that oral immunization with the recombinant L. plantarum provided an effective means for eliciting protective immune response against early ALV-J infection.In this study, a novel oral vaccine of recombinant  The J Subgroup Avian Leukosis Virus (ALV-J) is a subgroup of retrovirus that was first reported and isolated from broiler chickens in the United Kingdom in the early 1990s –5. This gp) with Mr of 85,000 (gp85) is encoded by the envelope (env) gene of ALV, and that the gp85 protein forms globular structures on the surface of the virus, and is closely associated with viral neutralization which determines the antigenicity of ALV-J , which can be used as a bacterial surface display element to immobilize the enzyme system on the surface of the cell membrane. In view of the characteristics of pgsA protein, it has been applied to various prokaryotic proteins on the surface display, in particular the lactic acid bacteria and other Gram-positive receptor strains (L. plantarum.Poly-γ-glutamate synthase A (pgsA) is a constituent protein of  strains . These fL. plantarum harboring pMG36e-pgsA-gp85 was constructed using genetic engineering technology, and then expressed on the surface of L. plantarum. The live recombinant L. plantarum was then used to orally vaccinate chickens. IgG and IgA antibodies against ALV-J, as well as viremia were detected to assess the effect of the recombinant L. plantarum.In the current study, the recombinant ALV-J-NX0101 strain, pMD18T-env recombinant vector (containing the gp85 gene), gp85-specific mouse monoclonal anti-body (MAb JE9), ALV-J antibody test kit, and ALV P27 antigen enzyme-linked immunosorbent assay (ELISA) test kits  were donated by Prof. Zhizhong Cui. The ALV IgA antibody test kit was purchased from Lanpai Biotechnology Company . The pgsA gene and purified gp85 protein were stored in our laboratory. The pMG36e expression vector was obtained commercially .The L. plantarum HQ542228 was purchased from China Center of Industrial Culture Collection (CICC) and grown anaerobically at 37°C, without agitation in MRS broth medium . The E. coli strains were cultured at 37°C with continuous shaking in Luria–Bertani (LB) medium .Hy-Line Brown layer chickens (1-day-old) were purchased from the Hylan Breeder Company  and housed in a specific-pathogen-free environment at the Laboratory Animal and Resources Facility, Shandong Agricultural University. The animals had free access to water and commercial standard pellet diet from Liuhe Jingwei Farming and Animal Husbandry Co., Ltd. . Each chicken was confirmed negative for the ALV-J antibody and virus by ALV-J-antibody ELISA initiation of the experiment. The experimental procedure was approved by the Animal Care and Use Committee of the Shandong Agricultural University and performed in accordance with animal welfare and ethics guidelines (SDAUA-2016-037).Bacillus subtilis pgsA genes, complete cds sequence published in GeneBank (Number AB016245.1) by primer 5.0 and synthesized in Sangon Biotech . The forward and the reverse primer of the pgsA gene was 5′-CGAGCTCGCGAACTGAGCTTTCATGAAAAG-3′ and 5′-CTAGTCTAGACTATGATCAATATCAAACGTCA-3′, containing the SacI and XbaI sites (underlined) respectively, with the plasmid T7-pgsBCA as the template. PCR amplification was performed as follows: 95°C for 5 min, 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 72 s, and 72°C for 5 min of final extension. The PCR product was confirmed by DNA sequencing. The gp85 gene was amplified using the forward primer (5′-TCATCTAGAGGGAGTTCATCTGTTG-3′) and the reverse primer (5′-TCCAAGCTTATTAGCGCCTGCTAC-3′) containing the XbaI and Hind III sites (underlined), respectively, with the pMD18T-env as the template. PCR amplification was performed as follows: 95°C for 5 min, 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1 min, and 72°C for 5 min of final extension. The PCR product was confirmed by DNA sequencing.The primers were designed according to the L. plantarum containing the plasmid of the pMG36e-pgsA-gp85 was identified via antibiotic selection and PCR experiments using the primers pgsA and gp85.Electroporation was performed according to the method of Josson et al. . BrieflySodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to analyze the expression of the target protein, and western blotting was developed using the gp85-specific mouse monoclonal antibody JE9 as the primary antibody. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  was used as the secondary antibody.L. plantarum cells were cultured in MRS broth overnight at 37°C. The cell pellets were sequentially incubated with gp85-specific mouse monoclonal antibody (1:800) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibodies . Finally, 3 × 104 cells were analyzed with a FACS Calibur platform  equipped with CellQuest software.For flow cytometry, L. plantarum group and natural L. plantarum group. Each group contained 40 chickens that were kept in separated isolators receiving filtered positive-pressure air. The recombinant L. plantarum group was immunized intragastrically with recombinant strains of L. plantarum (harboring plasmid pMG36e-pgsA-gp85), while the chickens in the control and negative control group were inoculated with PBS and natural L. plantarum, respectively. The suspensions of strains for administration were prepared as follows: recombinant strains cultured overnight were collected by centrifugation at 3,000 × g for 10 min, washed three times with sterile PBS, and then resuspended in sterile PBS to a concentration of 5 × 109 CFU/mL (400 μL/chicken). The control group and negative control group received equal doses of sterile PBS and natural L. plantarum. The immune protocol was administered on five consecutive days (days 1–5). A booster immunization was given between days 15 to 19, the second booster was given between days 29 to 33 and the third booster was given between days 43–47.The layer chickens  were randomly divided into three groups: control group, recombinant Blood samples were collected from the wing vein on Days 1 (before immunization), 7, 14, 28, 35, 42, and 49. Blood samples without additives were centrifuged and the serum was stored at −80°C for subsequent analysis.To obtain bile and intestinal lavages samples, 3 chickens were randomly selected from each group, and were killed on Day 1 (pre-immune), 7, 14, 28, 35, 42, and 49. Moreover, using a previously described method based on that of Wu and Russell , intesti450nm of sample–mean of OD450nm of negative control)]/[(mean of OD450nm of positive control–mean of OD450nm of negative control)]. The samples from each group were tested in three parallel trials, and the S/P values of any sample higher than 0.6 were considered as ALV-J antibody positive.The anti-ALV-J antibody in serum, bile and duodenal lavages were analyzed by ELISA. Briefly, the titers of IgG were tested using an ALV-IgG ELISA Kit  according to the requirements of the specification. The optical density (OD) of the IgG were measured at 450 nm and determined by the sample-to-positive (S/P) method following the formula below: S/P = [(mean of OD450nm of sample) + 0.15. The samples from each group were tested in three parallel trials, and the mean of OD450nm of each sample higher than CUT OFF were considered to be ALV-J antibody positive. In our study, the test was considered effective if the mean of the negative control wells was <0.10 and the mean of the positive control was >1.00.Furthermore, the commercial ALV-IgA antibody test kit  was used to detect the positive ratio of the anti-ALV-J IgA in serum, bile and duodenal lavages according to the manufacture's protocol. The OD of the IgA was measured at 450 nm and determined by the Critical value (CUT OFF) method following the formula below: CUT OFF = (mean of ODIndirect ELISA assay was used to assess the titers of anti-gp85 protein antibody. A 96-well ELISA plate was coated overnight with gp85 protein of appropriate concentration (4 μg/mL) and kept at 4°C. After blocking of the plates, the samples of serum, bile and duodenal lavages were used as the primary antibody and incubated at 37°C for 1 h. Then HRP-conjugated goat anti-chicken sIgA at a dilution of 1:2,000 (Sigma) at 37°C for 1 h was used to detect bound antibodies. Following washing the plate thice with PBS-0.05% Tween 20, tetramethylbenzidine  was added as the colorimetric substrate. Finally, the optical density (OD) was measured at 450 nm.3 TCID50 of the ALV-J NX0101 strain at Day 50. The plasma samples from the chickens were aseptically collected and then inoculated into the CEF cells in 24-wells plates. The cell supernatant of viremia was collected weekly to check for the presence of the virus using ALV P27 antigen ELISA test kits (IDEXX USA Inc.). The relative antigen titer level was determined by calculating the S/P ratio using the formula mentioned above. The samples from each group were tested in triplicate, and plasma samples with S/P ratios higher than 0.2 were considered virus-positive.ALV-J NX0101 strain was selected as the challenge virus and transfected into chicken embryo fibroblasts (CEF) for amplification and detection of virulence. The TCID50 of ALV-J was judged following the method described by Fadly . All of P-value was <0.05.Statistical analysis was performed using the SPSS software . Meanwhile, one-way ANOVA was used to identify the significant values. All values were expressed as mean ± standard error of the mean (SEM). All measurements were replicated three times. The differences were considered significant when the gp85 gene from the pMD18T-env recombinant vector (expected size 930 bp) is shown in pgsA gene from the T7-pgsBCA recombinant vector  is shown in The amplification of the pgsA-gp85 was expressed in L. plantarum, and confirmed by SDS-PAGE . Meanwhile, BW of chickens in the natural L. plantarum group were also higher than control, but without significant differences to the recombinant group.One week after vaccination, the BW of chickens in recombinant L. plantarum significantly triggered specific IgG and IgA antibodies against ALV-J, and enhanced the levels of IgG and sIgA compared to the control group  and then analyzed by ELISA. A low level of IgG titers was detected after the primary immunization. The IgG level increased slowly after the first booster immunization, and increased statistically after the second booster immunization on Day 21 . Peak IgG titers were reached after the third booster immunization on Day 35 (5 weeks). However, no significant specific antibodies were observed in the negative L. plantarum group until Day 49 the virus was challenged.The level of systemic IgG in serum increased significantly after oral inoculation as shown in L. plantarum group as shown in Samples were taken from three chickens in each group at random, at weekly intervals, from Day 1 (pre-immune) until Day 42 (6 weeks) and then analyzed by ELISA. The specific ALV-J IgA antibodies in bile, duodenal lavages and sera were detected during each test period . The sIgP < 0.01). The profile showed a steady increase before Day 21 (3 weeks) and achieved the highest level on the 35th day after the 3rd booster immunization . There were no significant differences between the L. plantarum group and PBS control group (P > 0.05).Results of the sIgA levels in bile  showed tP < 0.01). In addition, there was no significant difference between the L. plantarum group and PBS control group (P > 0.05). Therefore, these findings indicated that the recombinant pMG36e-pgsA-gp85 L. plantarum significantly enhanced the sIgA antibody response and produced local mucosal immune responses.The results of the sIgA level in duodenal lavages and sera  showed tL. plantarum+ALV-J and PBS+ALV-J groups were obviously higher than those in the recombinant L. plantarum+ALV-J group.Viremia were determined weekly after challenge until the 77th day, the positive viremia ratios were calculated, and the results were shown in Before 2005, ALV-J was widely recognized and reported in many regions of the world; it was predominantly prevalent in white feather broiler chickens , 25, 26.L. plantarum, a lactic acid bacteria, that can be used as a probiotic for animal gastrointestinal tracts, has many functions, such as maintenance of intestinal flora, enhancement of immunity, and promotion of nutrient absorption. L. plantarum can also act as a potential delivery vehicle for mucosal vaccines because it is generally regarded as safe and is able to protect antigens from premature disintegration and degradation in animal gastrointestinal tract  at 49, 63, 70, 77, 84 d, indicating that vaccination with recombinant L. plantarum can prevent body weight loss caused by ALV-J infection in chickens.Because ALV-J is able to cause growth retardation in infected chickens, body weight is considered as a valuable indicator to evaluate the protective effect of our established recombinant vaccine , 31. As L. plantarum vaccine. Efficient mucosal immunity is mediated predominately by secretory IgA . These results indicated that recombinant pMG36e-pgsA-gp85 L. plantarum effectively enhanced the sIgA antibody response and produced strong local mucosal immune responses (L. plantarum could act as an excellent mucosal adjuvant and vector of recombinant vaccines (IgA is the predominant antibody on the mucosal surface, as it is produced locally at a level that exceeds those of all other immunoglobulins , 36. Theesponses . Thus, Lvaccines .L. plantarum can induce high IgG in sera. IgG is the main antibody produced by the humoral immune response, and it plays an important role against infection, including neutralizing toxins and internal conditioning in body defense mechanisms (Moreover, mucosal delivery of vaccines should also elicit specific immunity in systemic lymphoid tissues because most infections that gain entrance through mucosal surfaces will become systemic –40, and chanisms . Some rechanisms , 43.L. plantarum group were significantly higher than those in the natural L. plantarum and control group from Day 7 to 49 (L. plantarum and control groups, after challenge, better protection was observed in the recombinant L. plantarum group against ALV-J.IgG is an important indicator for assessing the immunity efficiency of exogenous vaccines. In our study, results showed that IgG titers of the recombinant  7 to 49 . In addiL. plantarum, this can be administered orally to animals, tolerate gastric acidity, elicit both mucosal and systemic immune responses, and effectively alleviate viremia.In conclusion, our study demonstrated that the ALV-J gp85 protein was present on the surface of the non-pathogenic JL and YL designed the study. SW, YX, and NG contributed analytic tools. SW, YQ, and NG analyzed the data. JL, KL, and MS wrote the paper. DZ, YL, and MS revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli is associated with sequence type 131 (ST131). However, mechanisms of ST131 spread remain unclear. This study searched for proteins with amino acid substitutions specific for ST131 and used proteomics analysis to clarify ST131 characteristics. Five proteins had ST131-specific amino acid substitutions: uncharacterized protein YahO with E34A (m/z 7655); UPF0337 protein YjbJ with V59D, D60S and T63K (m/z 8351); uncharacterized protein YnfD with S106T (m/z 8448); and acid stress chaperone HdeA with Q92K and N94S (m/z 9714). Soluble cytochrome b562 (m/z 11783) showed seven amino acid substitutions, and the sequence differed between clade C of the pandemic clade and non-C. In silico analysis showed YahO protein-protein interaction with YjbJ, possibly related to biofilm formation. Although the function of soluble cytochrome b562 is electron transport of unknown function, its involvement in biofilm formation was predicted. HdeA was a gastric acid resistance-related protein. The function of YnfD was completely unclear. In conclusion, ST131-specific protein amino acid substitutions consisted mainly of a gastric acid resistance protein and proteins of unknown function (possibly involved in biofilm formation), which might be mechanisms for long-term colonization in the human intestinal tract.The global pandemic of ESBL-producing  Enterobacteriaceae have spread worldwide, mainly from Escherichia coli during the late 2000s4. Especially, ESBL E. coli have spread not only in hospitals but also in communities3. One of the causes of this spread is the global pandemic of E. coli sequence type (ST) 131 of multidrug-resistant E. coli6. ST131 is responsible for millions of global antimicrobial-resistant infections annually, for which the mortality rate is estimated to continue to exceed the current highest rate of malignant tumour mortality by 2050, if drug-resistant bacteria continue to increase without additional measures being taken7. The World Health Organization warned especially of multidrug-resistant Enterobacteriaceae such as E. coli in 20148. Therefore, new strategies to prevent its spread are required in the future. Infections caused by ST131 are considered to be more pathogenic and refractory than those caused by non-ST131 because ST131 carries more drug-resistance factors and urinary tract virulence factors than non-ST1311.Extended-spectrum β-lactamase (ESBL) and carbapenemase-producing multidrug-resistant fimH30 subclone and O25b serotype of ST131 were found to participate significantly in fluoroquinolone resistance and virulence factors11. Within the fimH30 subclone, the fimH30-Rx subset often carries blaCTX-M-1511. Therefore, these clones show a higher prevalence for resistance to cephalosporins9. Moreover, the current pandemic clones are strains classified into the C clade by whole genome analysis12. Particularly, clades C1 and C2 are prevalent worldwide. In addition, the C1-M27 subclade with blaCTX-M-27 became especially prominent after 200913. In this way, the population structure of ST131 has been explored extensively by many researchers. However, the reasons for the success of ST131 as an antimicrobial-resistant pandemic clone have not been clarified. The purpose of this study was to search for the proteins with specific amino acid substitutions for these clones using MALDI-TOF MS and identify them to clarify the characteristics of ST131 and its mechanisms of spread.Recently, the m/z 3236, 4176, 4857, 5381, 6827, 7655, 8351, 8448, 9710 and 11783 were detected as reproducible specific peaks of ST131. The peaks of m/z 4176, 4857, 8351 and 8448 were detected 3 out of 3 times, whereas those of m/z 3236, 5381, 6827, 7655, 9710 and 11783 were detected 2 out of 3 times. The peaks of m/z 3236, 4176, 4857, 7655, 8351, 9710 and 11783 were also detected as ST131-specific peaks in the relevant published references.The search for specific proteins and their amino acid substitutions of ST131 was performed by MALDI-TOF MS analysis. Table m/z 7655 peak was identified as uncharacterized protein YahO (Uniprot accession no. P75694) belonging to the Bhs/McbA family. The chain domain of the YahO protein was DUF1471, and its function is unknown. The m/z 8351 peak was identified as UPF0337 protein YjbJ (Uniprot accession no. P68206) belonging to the UPF0337 (CsbD) family. The domain of YjbJ protein was CsbD, and its function is unknown. The m/z 8448 peak was identified as uncharacterized protein YnfD (Uniprot accession no. P76172), and its family was unclear. The chain domain of YnfD protein was DUF1161, and its function is also unknown. The m/z 9710 peak was identified as acid stress chaperone HdeA (Uniprot accession no. P0AES9) belonging to the HdeA family. The domain was HdeA, and the function is stress response protein acid-resistance protein. The m/z 11783 peak was identified as soluble cytochrome b562 (Uniprot accession no. P0ABE7) belonging to the cytochrome b562 family. The mature chain domain was cytochrome_B562, and the function is electron-transport of unknown function. The m/z 3236 and 4857 peaks were multivalent ions of m/z 9710 of HdeA, and the m/z 4176 peak was a multivalent ion of m/z 8351 of YjbJ. The m/z 5381 and 6827 peaks were not identified in this study.Table Figure Escherichia coli K12 MG1655). The hdeA was included in the cluster mainly composed of gastric acid resistance-related proteins such as hdeB, gadA, gadB and gadC. Besides, it was predicted that yahO and yjbJ might indicate protein-protein interaction, and their function was unknown, including that of its neighbouring proteins. The ynfD was a node isolated from those clusters. The cybC was not detected in this database. Figure bsmA and bhsA have the DUF1471 domain as does YahO, and their function was related to biofilm formation. Furthermore, mcbA was colonic acid mucoidy stimulation protein, which was also a protein related to biofilm formation. Proteins common to the YjbJ domain were not searched. Figure cybC by the STRING database using E. coli CFT073 from a urinary tract infection. The cybC had an interaction with tomB, which is a biofilm formation regulator.Figure The purpose of this study was to search for and identify the proteins with amino acid substitutions specific for ST131 by proteomics analysis and to clarify the characteristics of ST131 to identify clear mechanisms for its spread. In this study, we found several proteins with the amino acid substitutions specific for ST131 and predicted their protein-protein interactions by bioinformatics using various web-based databases.E. coli collected from seven acute-care hospitals in the Kyoto and Shiga regions of Japan, the most specific peak of ST131 was m/z 9720, with a sensitivity of 97.0% and a specificity of 91.5%14. Also, in a study using 109 ESBL-producing E. coli collected from clinical settings and the environment in France, the peak was identified as m/z 971315. Moreover, in a study using 73 E. coli collected from different settings, geographic origins and isolation dates, several peaks of ST131 similar to those in the present study including m/z 9713 were detected16. However, no protein identification was performed in these previous studies. We also detected m/z 9710, which was similar to the peaks detected in these reports, and revealed it to be gastric acid resistance-related protein HdeA and found an amino acid substitution specific for ST131. HdeA localizes in the bacterial periplasmic space and exhibits acid resistance by expressing chaperone-like activity that suppresses denaturation of proteins under acidic conditions. Although it is unclear how this amino acid substitution affects the strains, ST131 might have high resistance to gastric acid that allows it to reach the human intestinal tract.MALDI-TOF MS typing of ST131 has been explored extensively by many researchers. In a study using 149 ESBL-producing m/z 7650 peak that is more specific than HdeA. We already found in our earlier study that YahO is a protein specific for ST13117. We predicted its function using the STRING and GeneMANIA databases, which predicted YahO to have protein-protein interaction with YjbJ, another protein we found, suggesting that they might be related to biofilm formation. Kudinha et al.18 and Clermont et al.19 both found that the prevalence of strains producing biofilms was greater among ST131 than among non-ST131 clinical strains. Besides, Eletsky et al.20 researched the function of the DUF1471 domains of Salmonella protein YahO, and they concluded that YahO is closely related to YcfR/BhsA, YbiM/McbA, YjfO/BsmA or YcfR, which are associated with a stress response and biofilm formation. This was also predicted in our study. In the future, it will be necessary to elucidate how this specific amino acid substitution of YahO and YjbJ affects biofilm formation. In addition, in the cybC sequence of soluble cytochrome b562, we found a specific sequence in the pandemic clade C. Moreover, cybC had an interaction with tomB, which is a biofilm formation regulator. These proteins possibly involved in biofilm formation may be related to the affinity of ST131 for humans.In this study, we found YahO protein showing a The characteristics of ST131 clarified in this study might allow a hypothesis of the mechanism of colonization in the human intestinal tract. We hypothesized that ST131 does not die when passing through the stomach because of its resistance to gastric acid, and when it reaches the intestinal tract, it survives for a long time by forming a biofilm in the intestinal tract. As this is only a hypothesis, it will be necessary to elucidate the influence of these amino acid substitutions in the future.fimH30 typing and the MALDI data, are consistent with those of other previous studies, we believe the findings are reliable. Second, as stated previously, although we clarified the difference in amino acid substitutions in various proteins between ST131 and non-ST131, the influence of these substitutions remains unclear. In the future, we will perform function analysis, such as pull-down and genome editing assays, of these proteins. Finally, although YnfD was identified as a protein with an amino acid substitution specific for ST131, its function could not be deduced at all.This study has several limitations. First, the ST131 and non-ST131 strains used in this study were collected only from various regions in Japan and not sites worldwide. However, the strains were collected from all over Japan and were not localized to specific regions. Because the strain characteristics, such as In conclusion, we found several proteins with amino acid substitutions specific for ST131. They consisted mainly of gastric acid resistance proteins and proteins with unknown function (but which were estimated to be biofilm formation proteins). These proteins might be associated with the mechanism of long-term colonization of ST131 in the human intestinal tract. In the future, by clarifying the functions of these proteins and their substitutions, the mechanism behind the worldwide pandemic of the ST131 might be further clarified.E. coli (97 strains of ST131 and 100 strains of non-ST131) isolated from clinical specimens collected at 24 clinical facilities in Japan between 2011 to 2013 were used. The 24 clinical facilities comprised 23 acute-care hospitals and 1 commercial laboratory located throughout Japan: 18 facilities from Western Japan and 6 facilities from Eastern Japan. These samples, which were derived from our previous study17 (n = 74) and the present study (n = 123), were kindly provided by the Association of Japan Community Healthcare Organization (JCHO) hospitals and the Study of Bacterial Resistance in the Kinki Region of Japan (SBRK) and were randomly extracted . In ST131 typing, ST131 was defined based on PCR detection of ST131-specific SNPs in the mdh and gyrB alleles21. In addition, the ST131 clade and subclade were defined based on multiplex PCR using seven specific SNP primers22. In O25b serotyping and fimH30 subclone typing, specific PCR detection based on each specific primer was performed23. In addition, fimH30-Rx was defined based on PCR detection of ybbW SNP typing24. In β-lactamase gene typing, strains were analysed to determine the presence of ESBL encoded by blaSHV, blaTEM, blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8-like and blaCTX-M-9-like26. In addition, PCR direct sequencing analysis was performed on blaSHV- and blaTEM-positive strains27.These strains were characterized by ST131 typing, ST131 clade typing, O25b serotyping, 17. The strains were cultured at 37 °C for 16-24 h using 5% sheep blood agar. We performed ethanol-formic acid protein extraction from grown colonies for preparation of the MALDI-TOF MS analysis and used Bruker Bacterial Test Standard  for calibration. MALDI-TOF MS analysis was performed using MALDI Biotyper . Spectra obtained by MALDI-TOF MS analysis were used for comparison of the spectrum of the ST131 with that of the non-ST131 using ClinProTools v2.2 , and peaks specific to ST131 were searched. We analysed spectra with Peak Statistic Calculation, which includes the Wilcoxon rank sum test. We analysed each isolate three times, and the peaks that were observed at least two of the three times in Peak Statistic Calculation were extracted as reproducible specific peaks of ST131. For the specific peaks judged to be significant by ClinProTools, peaks within the range of ±400 ppm were confirmed by FlexAnalysis v3.4 software  for each strain. The settings used for MALDI-TOF MS analysis were linear positive mode, 20-Hz laser frequency, 20-kV acceleration voltage, 18.5-kV IS2 voltage, 250-ns extraction delay, and 2000–20,000 m/z range.The method of MALDI-TOF MS analysis followed that of our previous study17. The strains with these specific peaks were incubated using 10 L LB broth, and then all pellets were collected after centrifugal separation. Next, to collect periplasmic proteins, the pellets were suspended with 30 mM Tris-HCl pH 8.0 with 20% sucrose on ice and incubated for 10 min at room temperature with slow mixing. Then, after centrifugal separation, the pellets underwent osmotic shock for 10 min using 5 mM MgSO4 solution on ice. After centrifugal separation, the supernatant was used as the periplasmic proteins fraction after being dialyzed against 20 mM Tris-HCl pH 8.0. The pellets were suspended with 20 mM Tris-HCl pH 8.0, and soluble cytoplasmic proteins were collected after sonication of the pellets for 15 min. After centrifugal separation, the supernatant was used as the soluble cytoplasmic proteins fraction after being dialyzed against 20 mM Tris-HCl pH 8.0. Then, each of the protein fractions was purified by ion-exchange chromatography using Macro-Prep diethylaminoethyl support  and reversed-phase HPLC using TSKgel ODS-100V . Finally, the gel obtained by tricine–SDS-PAGE with silver staining was analysed by LC-MS/MS using a High-Performance Liquid Chromatograph Ion-trap Time-of-Flight mass spectrometer  after in-gel digestion with trypsin of the target protein band was conducted. Trypsin digestion was performed using 10 µg/mL of sequencing grade modified trypsin , and the trypsin solution was incubated at 37 °C for 15 h. Spectra obtained from bottom-up proteomics were analysed using Mascot MS/MS Ion Search  to search and assign the obtained peptides to the SwissProt database. The identified proteins were investigated for protein name, localization, coding gene, family and domain, and currently known function using the Uniprot database (http://www.uniprot.org).The identification of the proteins with mass peaks specific to ST131 followed that of our previous studyhttp://pbil.univ-lyon1.fr/software/seaview) was used for sequence alignment and comparison. Also, the theoretical molecular weight calculated from the amino acid sequences was computed using the Compute pI/Mw tool (http://web.expasy.org/compute_pi/).From the amino acid sequencing identified by LC-MS/MS, the forward and reverse primers of each target protein were designed Table , and comhttps://string-db.org). Furthermore, the proteins whose functions were not predicted on STRING were predicted by investigating protein domain common proteins using GeneMANIA (https://genemania.org).The ST131 proteins predicted to have protein-protein interaction were identified by STRING database version 10.5 (Supplementary Table


Scientific Data 10.1038/s41597-019-0163-x, published online 21 August 2019Correction to: Following publication, it was found that due to a copying error a number of the values given in Table 4 of this Data Descriptor were incorrect. Table 4 has been corrected in both the HTML and PDF versions. The main text and underlying dataset remain unaltered.


Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. Our goal was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention. To do so, we performed next-generation RNA sequencing on doxycycline-treated spirochetes and treated spirochetes following regrowth, comparing them to untreated B. burgdorferi. A number of genes were perturbed and most of those which were statistically significant were down-regulated in the treated versus the untreated or treated/re-grown. Genes upregulated in the treated B. burgdorferi included a number of Erp genes and rplU, a 50S ribosomal protein. Among those genes associated with post-treatment regrowth were bba74 (Oms28), bba03, several peptide ABC transporters, ospA, ospB, ospC, dbpA and bba62. Studies are underway to determine if these same genes are perturbed in B. burgdorferi treated with doxycycline in a host environment.Recent studies have shown that  The efficacy of antibiotic treatment for Lyme disease has been a contentious issue among physicians and researchers . In partBorrelia burgdorferi persisters have been conducted using in vitro experimentation, and research on the development of drug tolerance in general is expanding  is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care-International. The Tulane University Institutional Animal Care and Use Committee approved all animal-related protocols, including the infection and sample collection from mice.Borrelia burgdorferi sensu stricto strain B31 clonal isolate 5A19  for this strain in a 5 day treatment protocol to be 2.5 μg/ml and the minimum bactericidal dose to be 50 μg/mL (B. burgdorferi cultures to be used for the bioassay were either not treated (0 μg/mL), treated with a concentration (10 μg/mL doxycycline) higher than the MIC but not greater than the minimum bactericidal concentration (MBC), or treated with the MBC. In all cases, BSK-II media was determined to be the optimal growth media for the experimental conditions after comparing growth rates of B. burgdorferi in BSK-II and BSK-H (data not shown). At day 5, the cultures were checked for motility by dark field microscopy (Low passage (p4 or p5) ate 5A19  was growate 5A19 , as descate 5A19 . Becauseate 5A19  they wer50 μg/mL . Thus, tcroscopy  , consisted of RNA from B. burgdorferi treated with 50 μg/mL doxycycline for 5 days. Group 3 (treated/re-grown), consisted of RNA from B. burgdorferi treated with 50 μg/mL doxycycline for 5 days, then allowed to regrow until the population reached the initial pre-treatment concentration of 5 × 107 cells/ml; this re-growth occurred between 10 and 12 days after the end of the treatment period.For RNA sequencing, duplicate escribed . Group 15B. burgdorferi derived from one of the three treatment groups was needle-inoculated into severe combined immune-deficient CB-17. SCID mice and C3H/HeN mice (Charles River Labs). Ear punch biopsies (2 mm) were taken on day 14 for all mice. On day 21, the mice were euthanized, and tissues including ear skin, heart, bladder, spleen and tibiotarsal joints were harvested. Tissues were used for culture in 5 mL of BSK-II and preserved in RNALaterTM to perform RT-PCR for both flaB and ospC genes. This was not performed in one experimental set of mice. The culture tubes were incubated in a tri-gas incubator as described above, and checked 2–3 times weekly, for up to 45 days, for signs of motility. The experiment was repeated three times, with a total of five control group SCID mice and C3H mice, a total of three 10 μg/mL doxycycline-treated-group SCID mice and C3H mice, and a total of seven 50 μg/mL-doxycycline-treated-group SCID mice and C3H mice. A summary of the experimental design is shown in A sample of 2 × 10B. burgdorferi following doxycycline treatment, Live/Dead BacLight® (Molecular Probes) staining was performed on untreated and 50 μg/mL-treated B. burgdorferi per the manufacturer’s instructions. Briefly, 1.0 μL of 1.67 mM SYTO-9, a green nucleic acid stain, and 1.0 μL of 1.67 mM propidium iodide, which will only stain cells with damaged cell membranes red, were thawed and mixed in equal proportions. A volume of 1.5 μL of the stain mixture was then added to 500 μL of B. burgdorferi suspended in phosphate buffered saline, pH 7.4 (Gibco), incubated in the dark for 15 min, then applied to a slide and coverslip for viewing and counting. The samples were viewed and counted in live/dead ratios using a fluorescent microscope. The excitation/emission maxima for SYTO-9 is 480/500 nm, and 490/635 nm for propidium iodide. Images captured were obtained using a Nuance FX®36 fluorescence microscope (Leica) and software, with an optimal emission filter range of 500–560 nm for SYTO-9, and 600–650 nm for propidium iodide. Results were calculated as percent viability, and reported as mean ± SD per group.To determine the proportion of live and dead flaB and ospC genes as described  using the RNeasy kit.Mouse tissues collected from the assessment of infectivity by antibiotic-treated spirochetes were subjected to RT-PCR on the escribed . B. burg primers . This in® One-step RT-PCR kit. Briefly, 0.4 nM of forward primer and 0.8 nM of the reverse primer were added to each reaction. The recommended cycling steps were used with annealing temperature up to 5°C below the melting temperature of each primer. Primers for amplification included: BB0778 = 5′TGTATGCACTGGTAGAAATA-3′(fwd) and 5′-TTTAACCTCTCCGTCTTTAT-3′(rev); BBA66 = 5′-GTTACAACCGTACCCGGAAATA-3′(fwd) and 5′-GCTGTCTTGGTTGACTAAAG-3′(rev); BBL39 = 5′-GGTGCTTGCAAGATTCATACTTC-3′(fwd) and 5′-CTCCTAAGTCTGCCCAGTTATT-3′(rev); BBN39 = 5′AGGAATCAGAAGACGAAGGATTAG-3′(fwd) and 5′-AACTTAGGCTCTTCGTAACCAG-3′(rev); BBJ09 = 5′-CAAGCTAAAGAGGCCGTAGAA-3′(fwd) and 5′-GCAAGCTTTGTATTGTTCGTAGT-3′(rev). Reactions were performed in a volume of 25 μl and 12 μl of each reaction was loaded onto the gel.A total of 10 ng RNA was added as template for the QiagenBorrelia burgdorferi cultures were grown to 5 × 107 cells/mL for the control, treated, and treated/regrown groups in 50 mL conical tubes in BSK-II media. For the control group, total RNA was extracted, while the treated and treated/regrown groups were treated with 50 μg/mL doxycycline for 5 days. On day 5, total RNA from the treated B. burgdorferi culture was extracted, while the treated/regrown B. burgdorferi culture was resuspended in doxycycline-free media, and monitored for regrowth. When the culture regrew to the initial concentration, total RNA was extracted. RNA extraction was done according to the Qiagen® RNEasy® Mini Kit manufacturer’s instructions. In all cases, RNA was kept in a –80° freezer when not being processed, and freeze-thaw cycles were kept to a minimum. After RNA extraction, the total RNA was assessed for concentration and purity with a ThermoFisher Scientific® NanoDropTM 2000 spectrophotometer. Samples with a total RNA concentration less than 300 ng/μL were not used in downstream steps. Next, for all groups, ribosomal RNA was depleted from the samples using Invitrogen Ribominus® kits that followed the manufacturer’s instructions. Finally, the RNA for all groups was then processed according to the Life Technologies Ion Total RNA-Seq kit v2 sample preparation instructions. RNA integrity was verified using a BioAnalyzer (Applied Biosystems) prior to RNASeq.B. burgdorferi genome FASTA and gene annotation GTF files were downloaded from PATRIC1 and the NCBI2 databases. After sequencing, the reference FASTA and annotation files were created from the B. burgdorferi chromosome and plasmids using a bash script and the sequenced samples were aligned using Bowtie2  test, included in the DESeq2 package, was used to assess statistical significance between experimental conditions.The  Bowtie2 . Read co Bowtie2 . A cutofThe database was constructed using a typical Drupal 7.x installation. A node was created for each gene, and using the pages module, html tables were created to display information for entire plasmids and the chromosome. Drupal indexed information for each node (webpage), which allowed the genes to be searchable using a search field. The data from the RNASeq experiment was also uploaded to the nodes, and designed to be searchable. The raw data are also available on the SRA (NCBI) database (accession # SUB5253665).B. burgdorferi were tested for infectivity in mice  mice because dormant or slow-growing spirochetes may not be able to evade the immune response of the host. As shown in B. burgdorferi, but positive organ cultures were apparent from both C3H and scid mice. One single positive culture was used to score them as positive. In the initial experiment, the untreated B. burgdorferi were grown for 5 days after reaching a density of 5 × 107 and had reached stationary phase prior to inoculation of mice. Thus, control mice (injected with 0 μg/mL-treated spirochetes) were not uniformly positive. For mice inoculated with 10 μg/mL-treated B. burgdorferi versus 0 μg/mL-treated B. burgdorferi, no significant differences in colonization were observed. The only somewhat significant difference was the number of scid mice infected with 50 μg/mL-treated B. burgdorferi versus 0 μg/mL-treated B. burgdorferi .Three conditions of  in mice  includin in mice . The untB. burgdorferi genes from ear and heart tissue of mice inoculated with antibiotic-treated spirochetes is shown in With respect to the molecular detection, mouse tissues were tested by RT-PCR for viability. Using several different targets, mice treated with 0 or 10 μg/mL were reliably positive, which correlated well with culture results. For those treated with 50 μg/mL of doxycycline, only one mouse was positive by RT-PCR and this result was found in the joint tissue by a nested set of primers. Amplification of B. burgdorferi during treatment with doxycycline and regrowth, three sample groups of B. burgdorferi were grown in BSK-II as untreated (G1), treated (G2), and treated/regrown (G3), where untreated was an untreated control, treated were spirochetes treated with the MBC of doxycycline, and regrown were spirochetes treated with the MBC of doxycycline then monitored for regrowth  and bbL39 (ErpN) were also subjected to RT-PCR to confirm the differential regulation  of the genes that were significantly differently expressed (p < 0.05) after treatment with doxycycline were found on lp54 (designated as BBA.._), most of which were down-regulated . The down-regulation of ospA was also observed; this may reflect a global down-regulation in gene expression and representative of the in vitro phenotype as ospA is down-regulated upon primary infection.To investigate the gene expression of gulation . Operon-egulated . This inAs expected, a large number of genes were down-regulated in the treated group versus the untreated group. These are associated with positive growth regulation, infection/virulence and metabolism. These included virulence factors, peptide and nucleic acid transporters, superoxide dismutase, and adenylate kinase.B. burgdorferi compared to those untreated may be of primary importance in establishing re-growth. In particular, bba03, bba74 (oms28), ospA, ospD, bba62, cspA and the oligopeptide permease protein genes oppF and oppD were up-regulated. Several of these gene products (outlined in the discussion) are associated with the habitation within the tick, and may therefore be indicators of dormancy, or exit from dormancy when expressed upon the blood meal  were much more abundant in the treated/re-grown compared to the untreated, indicating that they are associated with exiting dormancy rather than logarithmic growth, given that the growth phase was similar in the two groups.Several of these genes  for 6 days and then subjected to RNASeq, with comparison to the untreated control. Genes that were up-regulated by 2-fold or more were ascribed significant and the pathways affected were elucidated. In our study, we treated a slightly denser culture (5 × 107 versus 1 × 107) with doxycycline at the same dose (50 μg/ml) for 5 days. An aliquot of the treated cells was allowed to re-grow, such that we had 3 treatment groups. In addition, we performed RNASeq on duplicate samples and ascribed significance using both fold-change and p-values to account for variation between samples. Both studies shed light on the mechanisms that Bb may use to establish persistence and re-grow. The Feng study identified a large number of genes as upregulated following treatment, whereas we found that the vast majority of genes were down-regulated, likely owing to a global decline in transcription. In the Feng study, the ClpP protease was indicated as the most highly up-regulated gene in treated B. burgdorferi. It was up-regulated in our screen as well (0.43-fold), but not determined to be significant. The genes found to be significantly increased in treated versus control encoded several Erp proteins and a 50S ribosomal protein (bb0778). This was verified by standard RT-PCR, as shown in We have previously demonstrated that the persister cell phenotype appears to be generated stochastically and driven by slowed growth . In thisonducted , severalS. typhimurium, the toxins (TacT) add a post-translational modification (acetylation) to tRNA, which is reversed by a peptidyl tRNA hydrolase , originally thought to be a porin  and is subject to RpoS-mediated repression within the mammalian host. Meanwhile, ospA, ospB, and ospD appear to be regulated by the stress-response regulator bosR (bosR was shown to be up-regulated 0.325-fold following treatment and rpoS was up 0.386-fold; however, the p-values indicate that these were not significant perturbations. Interestingly, nine Erp proteins were found to be up-regulated by BosR (Analyses of tor bosR . These gtor bosR . In our  by BosR , so perhin vivo.The use of RNASeq to study adaptation , 2018 haPractices in the housing and care of animals conformed to the regulations and standards of the PHS Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. The Tulane National Primate Research Center is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care-International. The Institutional Animal Care and Use Committee of the Tulane National Primate Research Center approved all animal-related protocols, including the infection, treatment, and sample collection from mice.JC performed research experiments and data analysis, and contributed significantly to writing. ME conceived the experiments, participated in analyses, and contributed significantly to writing. NH, DM, and MC performed the experiments and proofed the manuscript. VC contributed to the experiments and data analysis. RS contributed to the data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.